[Histonet] RE: Tape Transfer

[gayle callis]

You Wrote (two messages) 
 
Dear Nele,
I hear of these problems very often. It would be worth your time looking for
any information on fresh frozen bone sections by Dr. Dodds, there should be
a number of papers he has published on good quality  bone sectioning
without the use of the slow, expensive and troublesome tape method.
 
Alan Bright
www.brightinstruments.com 
Sent from my BlackBerryR wireless device
 
-----Original Message-----
From: "Nele Degryse" <Nele.Degryse
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> UGent.be>
Sender: histonet-bounces
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t>
lists.utsouthwestern.edu
Date: Wed, 09 Feb 2011 09:30:44 
To: <histonet  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet>
<@t> lists.utsouthwestern.edu>
Subject: [Histonet] Tape transfer
 
Hello,
 
I'm trying to cut undecalcified bone (mouse knees) by using the tape  
transfer method.
The problem is that the area that I'm interested in (the joint with  
patella) is not transferred from the tape on my slide.
I've used slides with different coating (also those specially made for  
bone) and tried different thickness. None of that seems to make a  
difference.
Another problem is that my hand roller started to stick, so I cleaned  
it with 100% ethanol (as recommended) and now it sticks even more...
 
Can anybody give me some advice?
 
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***********************************

First, I totally disagree that using the "slow, expensive and troublesome"
tape method is a problem.  I took Dodd's workshop and then attempted cut
bone frozen sections in our lab using his methods which proved to be a
nightmare, taking hours to get one decent section IF we were lucky.   We
never "got lucky" in obtaining a  an intact bone section that was free from
folding and totally disrupted morphology.  If any method is troublesome, it
is the Dodd's method plus the expense of wasting time and nothing to show
for it.  The only thing I have used with any success was snap freezing bone
the way he taught us, but that has gone by the wayside due to the toxic
hexane fumes.    His way was a total failure in our laboratory and we bought
the Cryojane to get decent bone sections.  Cryojane requires practice, a bit
of patience,  fine tuning the little details for successful use and it is
not as slow as people think.   There are many labs who use it successfully
including ours.  

 

First of all, Nele did not provide any details on how the Cryojane was being
used other than the different polymer coated slides.  So questions follow:  

 

What was the cryostat chamber temperature?   Is this the same temperature of
the sample, knife and UV light source?    If the cryostat is older model,
have you actually checked the temperatures?  

 

Is the bone fixed in formalin before snap freezing?   And if the bone was
fixed, was it cryoprotected with at least 20 to 30% sucrose (this takes a
long time for NBF fixed bone)?   

 

What thicknesses did you try?  Thinner is often better than thicker.  

 

Is the d profile tungsten carbide knife perfectly sharp when cutting the
frozen bone sections?  This means a new edge for sectioning, and very
frequent reconditioning of the knife (DDK or Dorn and Hart sharpening
services).  Hopefully you have two knives.   

 

Have you tried multiple UV flashes?   Two to three flashes usually help keep
bone on the slide. 

 

Do you remove the tape INSIDE the cold cryostat environment, and at an angle
from one corner of slide across the section, slowly and gently. 

 

Have you tried orienting the bone differently?   Sometimes this helps,
particularly when removing the tape from the bone.    Is your mouse leg left
articulated so you section through the bent knee joint (into patella)?   

 

 

It sounds as though you have a temperature problem IF things are sticking.
When washing the roller, are you putting a lot of alcohol on the roller, or
simply wiping the roller with the alcohol.   Is the roller at the exact
cutting temperature of the sample before rolling the COLD tape onto the
block face?  

 

More details will help since we can't see a real time session of what you
are doing. 

 

Gayle M. Callis 

HTL/HT/MT(ASCP)